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J. Biol. Chem., Vol. 280, Issue 41, 34661-34666, October 14, 2005

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Chemerin Activation by Serine Proteases of the Coagulation, Fibrinolytic, and Inflammatory Cascades^*

Brian A. Zabel, Supported by National Institutes of Health Training Grant 5 T32 AI07290-151, Samantha J. Allen¶2, Paulina Kulig||, Jessica A. Allen, Joanna Cichy||3, Tracy M. Handel¶4, and Eugene C. Butcher

From the Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, ^¶Molecular and Cell Biology, University of California, Berkeley, California 94720, and ^||Faculty of Biotechnology, Jagiellonian University, Krakow 30-387, Poland

Received for publication, May 3, 2005 , and in revised form, June 22, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Proteases function at every level in host defense, from regulating vascular hemostasis and inflammation to mobilizing the "rapid responder" leukocytes of the immune system by regulating the activities of various chemoattractants. Recent studies implicate proteolysis in the activation of a ubiquitous plasma chemoattractant, chemerin, a ligand for the G-protein-coupled receptor CMKLR1 present on plasmacytoid dendritic cells and macrophages. To define the pathophysiologic triggers of chemerin activity, we evaluated the ability of serum- and inflammation-associated proteases to cleave chemerin and stimulate CMKLR1-mediated chemotaxis. We showed that serine proteases factor XIIa and plasmin of the coagulation and fibrinolytic cascades, elastase and cathepsin G released from activated neutrophil granules and mast cell tryptase are all potent activators of chemerin. Activation results from cleavage of the labile carboxyl terminus of the chemoattractant at any of several different sites. Activation of chemerin by the serine protease cascades that trigger rapid defenses in the body may direct CMKLR1-positive plasmacytoid dendritic cell and tissue macrophage recruitment to sterile sites of tissue damage, as well as trafficking to sites of infectious and allergic inflammation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A network of serine proteases regulates the primary response to injury and infection in the host. Serine proteases of the coagulation and fibrinolytic cascades mediate the homeostatic response to blood vessel injury. Kallikrein and factor XIIa process kininogens to generate bradykinin, a potent vasodilator that triggers increased vascular permeability during inflammation. Serine proteases termed convertases release multiple pathogen-neutralizing components of activated complement. Serine protease cascades also regulate the recruitment of phagocytic and antigen-presenting cells to sites of inflammation and tissue damage. The complement cascade, for example, releases active components C5a and C3a, potent attractants for many leukocytes, including neutrophils and monocytes (1, 2). Thus serine proteases are critical participants in rapid defense mechanisms in the body.

We and others have recently identified chemerin as a potent chemoattractant for cells expressing the G-protein-linked receptor chemokine-like receptor 1 (CMKLR1),⁵ also known as ChemR23 or DEZ (3–5). CMKLR1 is expressed in vitro by monocyte-derived macrophages and dendritic cells (3, 5, 6) and in vivo by circulating plasmacytoid dendritic cells (pDCs) (5) and tissue macrophages.⁶ pDCs are major producers of -interferons and can differentiate into antigen-presenting cells capable of triggering T effector or suppressor responses (7). Tissue macrophages have a major role as phagocytes but, similar to pDCs, are also implicated in bridging innate and adaptive immune responses and in regulating immunity in sterile versus infectious tissue injury (8). Importantly, chemerin is widely expressed and circulates in human plasma in an inactive state (5). Active forms of chemerin have been isolated from human ascites fluid, hemofiltrate, and serum and are characterized by apparent proteolytic cleavage of carboxyl-terminal amino acids present in the relatively inactive full-length pro-chemoattractant (3–5).

Here we have identified serine proteases as potent triggers of chemerin activation. Serum enzymes can activate recombinant chemerin, and this activity was blocked by selective serine protease inhibition. The clotting-associated serine proteases, factors XIIa and VIIa, and the fibrinolysis-associated serine proteases plasmin and plasminogen activators can all activate chemerin. Inflammatory cell-associated serine proteases, including neutrophil granule elastase and mast cell tryptase, are activators as well. Moreover, we found that several different cleavage sites, present in endogenous serum chemerin or generated during processing with specific enzymes, are sufficient for activation of this potent leukocyte attractant. These findings add chemerin to the list of innate immune mediators whose activity is critically regulated by serine protease cascades and implicate chemerin as an important link between these mechanisms of blood and tissue hemostasis and the recruitment of specialized CMKLR1-expressing "rapid responder" macrophages and dendritic cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In Vitro Transwell Chemotaxis—CMKLR1/L1.2 transfectants were generated as previously described (5). 5-µm pore Transwell inserts (Costar) were used for the migration assays. 100 µl of cells were added to the top well, and test samples were added to the bottom well in a 600-µl volume. The chemotaxis medium consisted of RPMI 1640 plus 10% fetal calf serum plus additives. Migration was assayed for 2 h at 37 °C, the inserts were removed, and the cells that had migrated through the filter to the lower chamber were counted by flow cytometry. 2.5 x 10⁵ cells/well were used as input, and the number of cells counted in 30 s was defined as the migration output. The results are reported as % input migration or % maximal migration.

Chemerin Expression and Purification Using Baculovirus—Chemerin with a carboxyl-terminal His₆ tag was cloned into pACGP67 (BD Biosciences) and transfected into Sf-9 cells. The expressed protein has the sequence NH₂-ADPELTEA... LPRSPHHHHHH-COOH, where the underlined residues are non-native. After viral amplification, chemerin was expressed by adding high titer virus to shaker flasks containing Hi-5 insect cells in Ex-cell 420 media (JRH Biosciences). After incubation for 2–3 days at 27.5 °C, the supernatant was harvested by centrifugation, filtered to 0.22 µm, and concentrated at 4 °C using a tangential flow concentrator (Filtron) with a 3-kDa cutoff filter. After a >100-fold buffer exchange into 50 mM HEPES, 0.3 M NaCl, pH 8.0, chemerin was purified by running the solution over nickel-nitrilotriacetic acid, SP-Sepharose (Amersham Biosciences) and C-18 reverse phase high pressure liquid chromatography columns (Vydac). The protein was lyophilized and checked for purity using electrospray mass spectrometry.

Chemerin Expression and Purification from Escherichia coli—Chemerin with a carboxyl-terminal His₆ tag (having the sequence NH₂-MELTEA...LPRSPHHHHHH-COOH, where the underlined residues are non-native) was expressed in TAP302 cells for 4 h at 37 °C. The cell pellets were spun down and detergent-solubilized by successive rounds of homogenization and spinning in the presence of 0.25% sodium deoxycholate. The insoluble inclusion body pellet was solubilized in a denaturing buffer (6 M guanidine HCl, 0.1 M sodium phosphate, 10 mM Tris, pH 8.0) and run over a nickel-nitrilotriacetic acid column using a decrease in pH to elute. The FoldIt screen (Hampton Research) was used to test suitable refolding conditions, and a modified version of buffer 11 was chosen as the best choice (50 mM HEPES, 0.3 M NaCl, 0.44 mM KCl, 2.2 mM MgCl₂, 2.2 mM CaCl₂, 550 mM L-arginine, 0.055% polyethylene glycol 8000, 1 mM reduced L-glutathione, 0.1 mM oxidized L-glutathione, pH 8.0). Chemerin was refolded by rapidly diluting into the refolding buffer at a final protein concentration of 0.1–0.2 mg/ml. After stirring for a few hours at 4 °C, the protein was rapidly diluted 20-fold further into column buffer (50 mM HEPES, 0.3 M NaCl, pH 8.0) and filtered. The protein was concentrated and purified using the tangential flow concentrator and chromatography, as described in the previous paragraph for baculovirus expression.

Serum—The Institutional Review Board at Stanford University approved all human subject protocols, and informed consent was obtained for all donations. Serum was stripped of heparin-binding proteins (including chemerin) by collecting the "flow-through" after passage over a heparin-Sepharose column. An amount of E. coli-expressed chemerin showing <5% input migration was incubated with an equivalent volume of serum or plasma for 5 min at 37 °C and then tested in a chemotaxis assay with CMKLR1/L1.2 transfectants.

Neutrophil-conditioned Media—Neutrophils were prepared from citrated peripheral blood obtained from healthy volunteers by density separation using Ficoll-Paque (Amersham Biosciences). The high density fraction containing neutrophils and erythrocytes was mixed (1:2 v/v) with 1% solution of polyvinyl alcohol in phosphate-buffered saline (Merck) and incubated for 20 min at room temperature. Neutrophils were collected from the upper phase and subjected to hypotonic lysis to remove contaminating red blood cells. Polymorphonuclear neutrophil (95% pure) were cultured for 20 h in serum-free RPMI 1640 medium. Conditioned media were collected, centrifuged, and normalized based on protein concentration, as determined by a BCA assay according to the manufacturer's specifications (Pierce). Conditioned media samples containing 14 µg of total protein were incubated for 5–10 min with recombinant chemerin.

View larger version (8K): [in this window] [in a new window]   FIGURE 1. Serum activation of chemerin is blocked by a serine protease inhibitor. Serum stripped of heparin-binding proteins to remove endogenous chemerin displayed no chemotactic activity but rapidly activated recombinant full-length pro-chemerin (titered to yield low initial activity, <5% CMKLR1 basal transfectant migration) during a 5-min incubation at 37 °C. Chemerin activation was abolished by pretreatment of the stripped serum with the serine protease inhibitor aprotinin but not by treatment with the cysteine protease inhibitor E-64. The mean of duplicate wells of a representative experiment (of three performed with similar results) is presented with range.

Mass Spectrometry—MALDI-TOF and electrospray mass spectrometry were performed by the Stanford Protein and Nucleic Acid Biotechnology Facility, the Protein Chemistry Core Facility, University of Columbia, and the Berkeley Protein Core Facility. Tryptic mass values were used in a Mascot search (www.matrixscience.com) of public peptide databases. PeptideCutter was used to predict the mass values of various chemerin isoforms and to predict tryptic chemerin fragments (www.expasy.org).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Serum Serine Proteases Activate Chemerin—Serum is significantly more potent than plasma in inducing the chemotaxis of CMKLR1-positive cells (5), suggesting that clotting might activate proteases capable of cleaving plasma chemerin to an active form. To test this hypothesis, we stripped serum of endogenous heparin-binding proteins (including chemerin) by passing it over a heparin column. This treatment effectively removed the endogenous serum chemotactic activity (Fig. 1). However, incubation of the undiluted stripped serum with recombinant full-length pro-chemerin dramatically enhanced the chemotactic activity of the protein. The triggering event was rapid, as chemerin chemotactic activity peaked within 5 min at 37 °C (data not shown). Aprotinin, a general serine protease inhibitor (but not the cysteine protease inhibitor E-64), blocked chemerin activation by stripped serum, indicating that serum serine proteases are required for chemerin activation. Furthermore, the canonical serine protease trypsin also activated chemerin, consistent with a direct activity of serine proteases on the attractant (TABLE ONE).

The serine proteases uPA and tPA cleave plasminogen to generate plasmin. Interestingly, these enzymes also activate chemerin (TABLE ONE). Although the enzyme concentrations required were higher (1000–10,000-fold) than their observed plasma zymogen levels (9), the uPA concentration was similar to the level required to cleave its primary physiologic target, plasminogen (11–13). Both plasminogen activators display increased abilities to activate plasminogen when in the bound state (14, 15), particularly tPA, which displays a kinetic acceleration of 50-fold in plasminogen activation in the presence of fibrin. Thus uPA and tPA may play a role in chemerin activation in vivo when concentrated on cell or matrix surfaces.

View larger version (8K): [in this window] [in a new window]   FIGURE 2. Neutrophil-conditioned media can activate chemerin. Human blood neutrophils were incubated for 20 h in serum-free RPMI 1640 medium. Conditioned media were collected, centrifuged, and normalized based on protein concentration, as determined by BCA assay. Conditioned media samples containing 14 µg of total protein were incubated for 5–10 min with recombinant chemerin. Proteolytic inhibitors were tested and media were incubated with 4 x 10-3 M pefabloc or 1 x 10-5 M E-64 for 30 min prior to the addition of chemerin, and chemotaxis was assessed with CMKLR1 transfectants. The mean from duplicate wells of a representative experiment (of two performed with similar results) is presented with range.

Neutrophil Granule Proteases Cathepsin G and Elastase Activate Chemerin—Neutrophils are recruited early to sites of acute inflammation and, when activated, release an array of enzymes and factors that regulate the inflammatory process, including the recruitment of other leukocytes (16). To determine whether neutrophils release proteases that activate chemerin, we initially incubated recombinant full-length chemerin with neutrophil-conditioned media. The media displayed potent gelatinolytic activity, indicating the release of neutrophil granule proteinases (data not shown). Neutrophil-conditioned medium, itself, had no chemotactic activity for CMKLR1 transfectants, but it displayed significant chemerin-activating ability (Fig. 2). Pefabloc, a general serine protease inhibitor, significantly blocked chemerin activation (Fig. 2), indicating that serine proteases released upon neutrophil degranulation activate chemerin. The serine proteases elastase and cathepsin G are major components of primary (azurophil) granules of neutrophils. Both proteinases generated active chemerin (TABLE ONE). Inhibition of neutrophil elastase and cathepsin G by their selective inhibitors, ₁-proteinase inhibitor and ₁-antichymotrypsin, respectively, abrogated chemerin activation (data not shown). Direct enzymatic activity, therefore, is required for the protease-induced generation of the active chemoattractant.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Identification of carboxyl-terminal chemerin-processing sites. A, chemerin was purified from human serum using heparin-Sepharose and gel filtration chromatography and PAGE separation, and MALDI-TOF analysis was performed following tryptic digest. Peptide fragments corresponding to chemerin are labeled with their mass values (a–g). Each of the labeled peptides was subjected to microsequencing by tandem mass spectrometry/mass spectrometry analysis to verify its origin from chemerin. B, the peptides corresponding to the chemerin fragments identified by MALDI-TOF are indicated by underlining and labeled a–g and represent 55% coverage of the secreted chemerin protein. The amino-terminal signal peptide is indicated by italics, as is the cleaved carboxyl-terminal peptide revealed by non-tryptic peptide (g). The vertical bars indicate trypsin cleavage sites, as predicted by PeptideCutter (www.expasy.org). C, controlled proteolysis of full-length recombinant pro-chemerin was carried out for 48 h at 37 °C using minimal amounts of the indicated proteases sufficient to yield functional chemotactic activities. The samples were then analyzed by electrospray or MALDI-TOF mass spectrometry. The amino acid sequence of the modified protein was extrapolated from the mass data obtained following proteolysis, and the resulting carboxyl-terminal sequence is displayed. The experimental mass data, the theoretical mass value of the predicted protein, and their Da difference is displayed for the indicated chemerin isoforms. The sequences of hemofiltrate and ascites forms are from Meder et al. (4) and Wittamer et al. (3), respectively.

Mast Cell Tryptase Is a Potent Chemerin Activator—Mast cell granules contain heparin, histamine, and numerous proteinases, the most abundant being the serine protease tryptase. Mast cell tryptase also circulates in the body, and although serum tryptase levels are quite low in healthy individuals (<1 x 10^-10 M), they can be 10–100-fold higher in patients undergoing anaphylactic reactions (21). Although low concentrations of tryptase (5.2 x 10^-11 M) did not activate chemerin, higher concentrations (5.2 x 10^-9 M) served as a potent activator (TABLE ONE).

Identifying the Carboxyl-terminal Processing Site for Endogenous Serum Chemerin—Wittamer et al. (3, 22) demonstrated that proteolytic cleavage in the carboxyl terminus of chemerin results in its activation. To identify the site of cleavage of serum chemerin, we isolated the active peptide from serum as described previously (5) and carried out mass spectrometric (MALDI-TOF) analysis of tryptic peptides. Peptides identified covered 55% of the protein sequence. In addition to peptides with canonical tryptic cleavage sites, one peptide displayed a mass value of 1669.7 Da, corresponding to a non-tryptic peptide comprising amino acids 141–155 from the carboxyl terminus of chemerin (Fig. 3, A and B). This peptide defines the carboxyl-terminal processing site of serum chemerin NH₂...FA FSKALPRS... COOH (Fig. 3, B and C). In corroboration, MALDI-TOF mass spectrometry of a chemerin-enriched fraction from human serum displayed a peak with a mass value of 15648.1 Da, within 5.2 Da of the predicted value of the truncated form above (15642.9 Da) (data not shown). This cleavage site is distinct from those defined previously by Meder et al. (4) from hemofiltrate and Wittamer et al. (3) from ascites (Fig. 3C) but has been recently reported by Busmann et al. (23) as a processed form present in Chinese hamster ovary cell supernatant.

Distinct Carboxyl-terminal Chemerin Cleavage Sites for Plasmin, Elastase, and Tryptase—To define the sites of chemerin cleavage by specific activating enzymes, we carried out controlled digestion of the recombinant full-length molecule to generate the active form(s) and used electrospray and MALDI-TOF mass spectrometry to define the cleavage products. This approach identified a single dominant plasmin carboxyl-terminal peptide cleavage site as NH₂... FAFSK ALPRS-COOH. This is a canonical serine protease (trypsin) cleavage site, as predicted by PeptideCutter software (www.expasy.org). Several carboxyl-terminal cleavage sites were observed following controlled activation with neutrophil elastase, consistent with its less discriminating activity and its preference for cleaving bonds adjacent to small, often hydrophobic residues. One of these is the same as that of the endogenously active chemerin in serum, NH₂... FA FSKALPRS... COOH. As summarized in Fig. 3C, mast cell tryptase also generated several distinct carboxyl-terminal cleavage products, including the serum form.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Multiple serine protease-mediated pathways triggered by injury or infection lead to chemerin activation. An interconnected network of serine proteases is activated upon tissue injury and/or infection, which triggers the primary protective response of hemostasis and inflammation in the host. Circulating pro-chemerin is rapidly activated by serine proteases triggered at the site of tissue damage. The plasma proteases plasmin, factors XIIa and VIIa, uPA, tPA, as well as the inflammatory proteases cathepsin G and elastase (released by infiltrating neutrophils) or tryptase (released by resident activated mast cells), are all potent activators of chemerin. Circulating pDCs may be specifically recruited to sites enriched in active chemerin via CMKLR1, where they can differentiate into pro-inflammatory effector-generating antigen-presenting cells or into immune-suppressive dendritic cells. Other rapid responder CMKLR1-positive cells, including tissue macrophages, may also be recruited to sites of tissue damage via chemerin activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

With the data presented here, three endogenously active human chemerin isoforms have been isolated, all with different carboxyl-terminal truncations. We have shown in Fig. 4C that certain serine proteases can directly generate the forms reported by Wittamer et al. (3) in ascites or observed by us in serum. Our results indicate that the inflammatory cell-associated serine proteases, elastase and tryptase, can generate additional active forms, each sharing the essential feature of cleavage of carboxyl-terminal, apparently inhibitory amino acids. Whether each of the several distinct cleavage products generated by tryptase and elastase are equally active as CMKLR1 ligands remains to be determined.

The potential for complex orchestration of leukocyte attraction by serine proteases during local tissue responses is well illustrated by the growing appreciation for the many roles of plasmin in this regard. Plasmin is, itself, a chemoattractant for monocytes (25). Plasmin cleavage of complement component C3 generates the split product C3a, which is a potent chemoattractant for eosinophils and mast cells (26). Plasmin-mediated proteolysis activates the pleiotropic cytokine transforming growth factor- (27), as well as another abundant plasma chemoattractant, CCL14 (HCC-1 or hemofiltrate CC chemokine 1; unrelated to chemerin), which attracts T cells, eosinophils, and monocytes (11, 28). Chemerin thus joins a growing family of immunomodulatory chemoattractants and cytokines regulated by plasmin and the fibrinolytic cascade.

Interestingly, the chemerin receptor CMKLR1 is a member of a subfamily of leukocyte G-protein-coupled receptors that includes chemoattractant receptors for a number of proteolytically activated or regulated ligands. The receptor for C3a, for example, is closely related to CMKLR1, as is the receptor for the complement component C5a (29), another blood protein that is proteolytically activated and involved in rapid responses linking innate and adaptive immunity. FPRL1, another member of the receptor subfamily, is expressed on neutrophils and acts as a chemoattractant receptor for human cathelicidin LL-37 (30), which is also generated by proteolytic processing from an inactive precursor (31). A hallmark of this chemoattractant receptor subfamily thus appears to be an affinity for serine-protease-activated ligands involved in the rapid host defense or response to injury.

By analogy with the processing of other proteolytically regulated mediators of acute inflammatory responses (see below), additional proteolytic enzymes, such as the abundant serum carboxypeptidase N (32) or the inducible carboxypeptidase R (also known as thrombin-activable fibrinolysis inhibitor or TAFI, which is activated locally by thrombin, plasmin, or neutrophil elastase (33–35)) could also play a role in the generation of different chemerin forms. These carboxypeptidases preferentially remove carboxyl-terminal lysine or arginine residues and thus could, by cleaving the terminal lysine from plasmin-activated chemerin, provide an alternative path for generating the endogenous ascites form reported by Wittamer et al. (3). Carboxypeptidase N and R remove the carboxyl-terminal arginine from C5a and C3a, which in this case generates "desarg" forms that are inactive (36). Thus these chemoattractants are first activated by serine proteases and then inactivated by further protease processing. A multistep, serine protease-initiated proteolytic cascade may also regulate chemerin activity in vivo, with carboxypeptidases or other enzymes participating in the processing of different active forms and in the eventual inactivation of the attractant. Furthermore, because the serum from which we isolated chemerin was generated from whole blood and thus contained red blood cells and leukocytes, it is quite likely that chemerin was exposed to additional proteases beyond those associated with the coagulation/fibrinolytic pathways (as would of course be the case in vivo as well). Cell-associated proteases, or proteases released upon hemolysis, may have contributed to chemerin processing, which may explain the discrepancy between the plasmin-cleaved isoform and the observed serum isoform.

Extracellular proteolytic processing also modifies the chemotactic activities of many chemoattractants of the chemokine family. As opposed to the activating effects of serine protease cleavage of chemerin, however, chemokine cleavage often results in inactivation or even antagonistic behavior. For example, CXCL12 cleavage by the membrane-bound protease CD26 (dipeptidylpeptidase IV) generates a CXCR4 antagonist (37). CD26 cleavage of CCL5 RANTES reduces its activity to attract CCR1-expressing cells (38). Gelatinase A cleavage of CCL7 (MCP3) generates a CCR5 antagonist (39). Cleavage of basic platelet protein by cathepsin G, however, generates CXCL7 (NAP2), a potent neutrophil chemoattractant that acts through CXCR2 (40). Thus the extracellular processing of chemoattractants represents a potentially critical regulatory mechanism for the physiologic recruitment of leukocytes.

In previous studies, we found that CMKLR1 is highly expressed by circulating pDCs in human blood and that this distinguishes pDCs from myeloid (mDC), the other major class of dendritic cell, thus offering a mechanism for their differential trafficking (5). pDCs are versatile cells that can act as tolerogenic or pro-inflammatory antigen-presenting cells. The effector functions of pDCs depend on the secondary signals they receive after recruitment to a tissue site. When they encounter toll receptor ligands (e.g. bacterial CpG or viral particles), pDCs develop into pro-inflammatory interferon -secreting cells that also acquire and process antigen. Pro-inflammatory pDCs down-regulate CMKLR1 (5) and up-regulate responsiveness to CCR7 ligands (41, 42), thereby permitting their migration to lymph nodes via afferent lymphatics and subsequent antigen presentation to T cells. On the other hand, pDCs stimulated by CD40L in the absence of toll receptor ligands develop into suppressor-type pDCs, which induce development of CD8 T regulatory cells (43). This capacity to suppress inappropriate immune responses when only internal tissue damage or sterile bleeding is involved may be one of the key roles of these cells, and in this context, their rapid deployment in response to coagulation or fibrinolytic enzyme activation of chemerin would provide a means to prevent inflammatory exacerbation of local tissue injury.

Other cells can express CMKLR1 as well. For example, Wittamer et al. (3) demonstrate that monocyte-derived macrophages can express CMKLR1 in vitro. In recent mouse studies, we have found F4/80+ cells of the macrophage lineage can also express the receptor and respond actively to chemerin.⁶ Indeed, tissue macrophages in human ascites fluid express CMKLR1 as well,⁶ a finding that suggests the potential for additional roles for chemerin in recruiting phagocytes and immune regulatory cells to sites of protease activity.

Interestingly, tissue macrophages are, similar to pDCs, flexible regulators of local immune responses capable of becoming potent interleukin-10-producing immunosuppressors or interferon -producing cells contributing to a pro-inflammatory milieu (44). Thus, it is attractive to hypothesize that the primary role of chemerin is to attract early responder "immunointerpreters" wherever there is a vascular breech or damage to cells; the role of the interpreters (pDCs and tissue macrophages) is to evaluate local conditions (perhaps a bit of blood, or invasion by bacteria, etc.) and to rapidly establish the appropriate suppressive or pro-inflammatory response.

In conclusion, we have shown that several serine proteases of the hemostatic and inflammatory cascades activate the widespread pro-attractant chemerin, providing a mechanism for rapid responder recruitment of CMKLR1-positive cells to sites of tissue damage or cell injury (Fig. 4). This model reinforces the concept of an organized and unified host response to injury and/or infection based on an interconnected network of serine proteases and provides a mechanism for recruitment of specialized immunointerpretive and immunoregulatory pDCs and CMKLR1-positive macrophages.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants AI-59635, AI-37832, AI-47822, and GM-37734, Specialized Center of Research Grant HL-67674, Digestive Disease Center Grant DK56339, and a Merit Award from the Veterans Administration (to E. C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Supported by a postdoctoral fellowship from the Cancer Research Institute, New York. Present address: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093

³ Supported by Jagiellonian University grants and a Fulbright fellowship.

⁴ Supported by grants from the National Institutes of Health (AI37113-09), the University of California Discovery Program (Bio03-10367), and the University of California AIDS Program (1D03-B-005). Present address: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.

¹ To whom correspondence should be addressed: Dept. of Pathology (5234), Stanford University Medical Ctr., Stanford, CA 94305. Tel.: 650-493-5000 (ext. 63132); Fax: 650-858-3986; E-mail: bazabel{at}alum.mit.edu.

⁵ The abbreviations used are: CMKLR1, chemokine-like receptor 1; pDC, plasmacytoid dendritic cell; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator.

⁶ B. A. Zabel, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank A. Bankovich for his expert technical assistance and advice with the chemerin purification from serum and L. Zuniga and J. Zabel for helpful discussions.

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